1. Field of the Invention
The present invention relates to a fluorescent biosensor that functions by a novel Quencher-Tether-Ligand (QTL) mechanism. In particular, the present invention relates to improving the polymer-QTL approach by co-locating the fluorescent polymer (or polymer ensemble, including self-assembled polymers) and a receptor for the QTL bioconjugate and target analyte on the same solid support.
2. Discussion of the Background
The polymer-QTL (Quencher-Tether-Ligand) approach is a single-step, instantaneous, homogeneous assay where the amplification step is intrinsic to the fluorescent polymer. The polymer-QTL approach provides a system for effective sensing of biological agents by observing fluorescence changes. The key scientific basis is the amplification of quenching of fluorescence that can be obtained with certain charged conjugated polymers and small molecule quenchers. In addition, the process is uniquely simple because there are no reagents.
In the “biosensor” mode, the QTL approach functions by having a fluorescent polymer quenched by a specially constructed “quencher-tether-ligand” (QTL) unit as shown in the diagram set forth in FIG. 1. Addition of an analyte containing a biological receptor specific to the ligand is expected to remove the QTL conjugate from the polymer which results in a “turning on” of the polymer fluorescence. A fluorescent polyelectrolyte-based superquenching assay has been shown to offer several advantages over conventional small molecule based fluorescence assays. For example, conjugated polyelectrolytes, dye-pendant polyelectrolytes, etc. can “harvest” light effectively both by absorption and by superquenching (1–5). The enhanced absorbing power of the polymers is indicated by the observation that even sub nanomolar solutions of some of these materials are visibly colored. The fluorescence of these polymers can be detected at even lower concentrations. Superquenching occurs in the presence of small molecules capable of serving as electron transfer or energy transfer quenchers to the polymer or one of its repeat units.
The “Stern-Volmer” quenching constants (KSV) for these polymers have been shown to be as high as 108–109 M−1, and it is anticipated that values as high as 1011 M−1 may be attainable (6). Such high values for KSV toward quenchers oppositely charged to the polyelectrolyte are initiated by strong nonspecific binding between the quencher and the polyelectrolyte. Subsequent amplified quenching occurs due to a combination of excitonic delocalization and energy migration to the “trapsite” where the quencher is in close proximity with the polymer.
It has also been shown that enhanced superquenching may be obtained when the polymers are adsorbed onto charged supports including surfaces, polymer microspheres, and inorganic nanoparticles (7,8). Superquenching has also been observed in the same supported formats for monomers or small oligomers self-assembled into “virtual” polymers. Polymer (and “virtual” polymer) superquenching has been adapted to biosensing by constructing QTL conjugates containing a potential superquenching component (Q) tethered (T) to a bioreceptor (L) or ligand for a specific biomolecule (1).
A fluorescence based assay is realized when the QTL conjugate is used to quench the polymer either in solution or in supported formats at solution-solid or solution-particle interfaces (1,7,8). For example, fluorescent polyelectrolytes, including conjugated and J-aggregate polymers, can be used for sensitive biodetection and bioassays in solution formats. The basis of this detection is the combination of the “superquenching” sensitivity of these molecules to quenchers of opposite or neutral charges with the synthesis of a quencher-recognition conjugate (e.g., a QTL molecule). In the original formulation, the QTL conjugate quenches the polymer ensemble by nonspecific binding. Addition of a target bioagent capable of binding with the L component of the QTL conjugate results in a removal of the QTL conjugate from the polymer and a turning on of the polymer fluorescence.
A fluorescence turn off (or modulation) assay has also been developed based on polymer superquenching (5). In this case, the target molecule is a bioagent L, or L′, corresponding to the L component of the QTL conjugate, and the receptor is a biomolecule that strongly associates with L, L′ or the QTL conjugate. One example is a direct competition assay in which L (or L′) in unknown amount is allowed to compete with the QTL conjugate for the binding sites of a measured amount of the receptor. The polymer fluorescence is quenched by non-bound QTL to an extent depending on the amount of L (or L′) present. In another example, the QTL conjugate is preassociated with the receptor; when all of the QTL conjugates are associated with the receptor sites, no quenching is observed. Addition of L (or L′) to the sample results in the release of the QTL conjugate with concomitant quenching of the polymer fluorescence.
All of the above assay formats depend on nonspecific quenching of the polymer fluorescence by association of the QTL conjugate with the polymer. A complication with these assays is the competing nonspecific interactions of other components of the assay sample with either the polymer, the QTL conjugate, or both, which result in a modulation of the quenching. In the present invention, modifications of the polymer superquenching allow the construction of improved assays which overcome these effects and provide for a more versatile and robust sensor.